School of Physiology (ETDs)

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Browsing School of Physiology (ETDs) by Author "Amoni, Joel Ikechukwu"

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    Understanding the in vitro effect of TNF-α and hyperglycaemia on endothelial activation
    (University of the Witwatersrand, Johannesburg, 2023) Amoni, Joel Ikechukwu; Millen, A; Gunter, S
    Background. Inflammation is one of the main underlying mechanisms in the development of cardiovascular disease (CVD). Indeed, in diseases characterised by high-grade systemicinflammation and in comorbid conditions, such as diabetes mellitus, it has been suggested that inflammation, at least in part, contribute to the increased risk of CVD. One of the earliest signs of inflammation-induced CVD is endothelial dysfunction. However, whether inflammation promotes endothelial dysfunction via the same signalling pathways in different pathological conditions characterised by systemic inflammation is not well understood. Inflammation- induced aberrant expression of microRNA (miRNA), small non-coding RNAs that function to regulate gene expression post-transcriptionally, have been linked to impaired endothelial function. However, the mechanisms whereby miRNAs may mediate endothelial activation requires further investigation. Therefore, the aim of this study was to investigate the molecular mechanisms involved in the regulation of endothelial function in different models of inflammation. Methods. EA.hy926 immortalized endothelial cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) + HAM’s F12 nutrient mix supplemented with 10% foetal bovine serum (FBS). EA.hy926 cells were exposed to tumour necrosis factor-alpha (TNF-α) at a concentration of 10ng/ml for 24 hours to induce an inflammatory response while the controls were cells exposed to plain media for 24 hours. EA.hy926 cells were also exposed to either 5mM or 30mM glucose for 72 hours, as a model of glycemia-induced inflammation, while the control cells were exposed to plain media for 72 hours. Total RNA was extracted from the cell pellets and subsequently reverse transcribed to miRNA cDNA and mRNA cDNA. Quantitative real time PCR was used to determine the relative expression of interleukin-6 (IL-6), vascular cell adhesion molecule 1 (VCAM-1), miRNA-155-5p, endothelial nitric oxide synthase (eNOS) and superoxide dismutase 2 (SOD-2). Additionally, an ELISA assay was used to determine the ratio of phosphorylated p65/total p65 in cells exposed to TNF-α (10ng/ml for 24 hours), glucose (30mM for 72 hours) and plain media controls. Results. Compared to control cells, the relative mRNA expression of the inflammatory marker IL-6 was significantly increased in the cells exposed to TNF-α (p=0.002) and 5mM (p=0.002) and 30mM (p = 0.0001) glucose, respectively. In addition, the relative mRNA expression of VCAM-1 was increased in the cells exposed to TNF-α (p <0.0001) and 30mM glucose (p =0.03) when compared to their respective controls. Interestingly, miRNA-155-5p expression was also increased in the cells exposed to TNF-α (p= 0.04), 5 mM glucose (p = 0.007) and 30 mM glucose (p = 0.02). Exposure to TNF-α, and 5 mM and 30 mM glucose resulted in increased eNOS expression compared to the control cells (p = 0.04, p=0.002 and p = 0.0002, respectively). The ratio of phosphorylated-to-total NF-κB p65 were not different in either the TNF-α exposed or glucose exposed cells compared to control cells (all p>0.05). Conclusion. These findings suggest that TNF-α and hyperglycaemia exposure resulted in endothelial dysfunction. However, hyperglycaemia caused much greater oxidative stress (increased eNOS) most likely due to glucose scavenging of nitric oxide (NO). This suggests that the underlying mechanisms of endothelial dysfunction occurring due to hyperglycaemia and inflammation may be driven by different mechanisms. This study highlights the need for further investigation into the mechanisms whereby miRNA-155-5p regulate endothelial dysfunction